1. Field of the Invention
The invention is directed to compounds that are specific inhibitors of PKC delta, and, in particular, to methods and compositions as therapeutical treatments, and as diagnostics to treat or prevent disorders such as cancers.
2. Background of the Invention
Targeting cancer therapeutics towards specific mutations or abnormalities in tumor cells not found in normal tissues has the potential advantages of high selectivity for the tumor and correspondingly low secondary toxicities. At least 30% of all human malignancies display activating mutations in the RAS genes, and perhaps another 60% display other activating mutations in, or over-activity of, p21Ras signaling pathways. It was previously reported that aberrant activation of Ras produces an absolute dependency upon PKC delta-mediated survival pathways (Xia S, Forman L W, & Faller D V 2007 Protein Kinase C{delta} is required for survival of cells expressing activated p21RAS. J Biol. Chem. 282 13199-13210; Xia S, Chen Z, Forman L W, & Faller D V 2009 PKCdelta survival signaling in cells containing an activated p21Ras protein requires PDKI. Cell Signal. 21 502-508). Over-activity of p21Ras signaling therefore sensitizes tumor cells to apoptosis induced by suppression of PKC delta, whereas suppression of PKC delta is not toxic to cells with normal levels of p21Ras activity or signaling (Chen C Y & Faller D V 1995 Direction of p21Ras-generated signals towards cell growth or apoptosis is determined by protein kinase C and Bcl-2. Oncogene 111487-1498; Xia S, Forman L W, & Faller D V 2007 Protein Kinase C{delta} is required for survival of cells expressing activated p21RAS. J Biol. Chem. 282 13199-13210; Chen C Y & Faller D V 1996 Phosphorylation of Bcl-2 protein and association with p21(Ras) in Ras-induced apoptosis. J. Biol. Chem. 271 2376-2379; Chen C Y, Liou J, Forman L W, & Faller D V 1998a Correlation of genetic instability and apoptosis in the presence of oncogenic Ki-Ras. Cell Death Differentiation. 5 984-995; Chen C Y, Liou J, Forman L W, & Faller D V 1998b Differential regulation of discrete apoptotic pathways by Ras. J. Biol. Chem. 273 16700-16709; Chen C Y, Juo P, Liou J, Yu Q, Blenis J, & Faller D V 2001 Activation of FADD and Caspase 8 in Ras-mediated apoptosis. Cell Growth Differ. 12 297-306; Liou J S, Chen C Y, Chen J S, & Faller D V 2000 Oncogenic Ras mediates apoptosis in response to protein kinase C inhibition through the generation of reactive oxygen species. J. Biol. Chem. 275 39001-39011; Liou J S, Chen J-C, & Faller D V 2004 Characterization of p21Ras-mediated apoptosis induced by Protein Kinase C inhibition and application to human tumor cell lines. J. Cell Physiol. 198 277-294). This tumor-susceptibility designated “Ras-mediated apoptosis” can be exploited for specific targeted cancer therapeutics.
Bronchopulmonary, gastrointestinal and pancreatic neuroendocrine tumors are rare tumors originating from neuroendocrine tissues (Oberg K 1999 Neuroendocrine gastrointestinal tumors—a condensed overview of diagnosis and treatment. Ann. Oneal. 10 Suppl 2:S3-8. S3-S8). Clinical symptoms are often caused by the production of hormonally-active substances by the tumor such as serotonin, gastrin, insulin, vasoactive intestinal peptide, pancreatic polypeptide, or substanceP. Chromogranin A is produced by 80-100% of neuroendocrine tumors and serves as a reliable biochemical marker. The disease can be cured by early surgery, but the vast majority of tumors have metastases at the time of diagnosis, which makes palliation the cornerstone of management. Debulking surgery, liver artery embolization, and chemotherapy aim at tumor mass reduction, whereas somatostatin analogues and IFN are used for control of symptoms (Arnold R, Simon B, & Wied M 2000 Treatment of neuroendocrine GEP tumours with somatostatin analogues: a review. Digestion. 62 Suppl1 84-91; Frank M, Klose K J, Wied M, Ishaque N, Schade-Brittinger C, & Arnold R 1999 Combination therapy with octreotide and alpha-interferon: effect on tumor growth in metastatic endocrine gastroenteropancreatic tumors. Am. J. Gastroenterol. 94 1381-1387). Radioactively-labeled somatostatin analogues have been used in trials, with response rates 30% (Arnold R, Wied M, & Behr T H 2002 Somatostatin analogues in the treatment of endocrine tumors of the gastrointestinal tract. Expert. Opin. Pharmacother. 3 643-656).
Response rates of cytoreductive approaches to such cancers are generally below 60%, however, and their use has limited utility because long-term responses are not maintained (Oberg K 2001 Chemotherapy and biotherapy in the treatment of neuroendocrine tumours. Ann. Oncol. 12 Suppl2: S111-4.). Accordingly, new and more effective approaches are therefore needed in the treatment of neuroendocrine malignancies.
Carcinoid and other neuroendocrine tumors of the gastrointestinal tract share a number of the same genetic abnormalities (deletions and mutations) as adenocarcinomas (Leotlela P D, Jauch A, Holtgreve-Grez H, & Thakker R V 2003 Genetics of neuroendocrine and carcinoid tumours. Endocr. Relat Cancer. 10 437-450; Leotlela et al. 2003; Arber N, Neugut A I, Weinstein I B, & Holt P 1997 Molecular genetics of small bowel cancer. Cancer Epidemiol. Biomarkers Prev. 6 745-748). These abnormalities include activation of Ras directly by mutations, indirectly by loss of Ras-regulatory proteins such as NF-1, or via constitutive activation of downstream effector pathways of Ras, such as PI3K and Raf/MAP kinase. For example, activation of H-Ras and Ki-Ras are detected in a significant fraction of carcinoid and other gastrointestinal tumors (65% and 10%, respectively) (Liedke M, Karnbach C, Kalinin V, Herbst B, Frilling A, & Broelsch C E 1998 [Detection of H-ras and K-ras in tumors of gastrointestinal-pancreatic system]. Langenbecks Arch. Chir Suppl Kongressbd. 115 255-259; Maitra A, Krueger J E, Tascilar M, Offerhaus G J, Angeles-Angeles A, Klimstra D S, Hruban R H, & Albores-Saavedra J 2000 Carcinoid tumors of the extrahepatic bile ducts: a study of seven cases. Am. J. Surg. Pathol. 24 1501-1510). Ras can also be activated in carcinoid and other neuroendocrine by either point mutation or loss of regulators of Ras, such as RassF1A or NF-1 (Liu L, Broaddus R R, Yao J C, Xie S, White J A, Wu T T, Hamilton S R, & Rashid A 2005 Epigenetic alterations in neuroendocrine tumors: methylation of RAS-association domain family 1, isoform A and p16 genes are associated with metastasis. Mod. Pathol. 18 1632-1640; Stancu M, Wu T T, Wallace C, Houlihan P S, Hamilton S R, & Rashid A 2003; Genetic alterations in goblet cell carcinoids of the vermiform appendix and comparison with gastrointestinal carcinoid tumors. Mod. Pathol. 16 1189-1198; Bausch B, Borozdin W, Mautner V F, Hoffmann M M, Boehm D, Robledo M, Cascon A, Harenberg T, Schiavi F, Pawlu C, et al. 2007 Germline NF1 mutational spectra and loss-of-heterozygosity analyses in patients with pheochromocytoma and neurofibromatosis type 1. J. Clin. Endocrinol. Metab. 92 2784-2792). The Raf/mitogen-activated protein kinase (Raf/MAP kinase), or the MAP kinases directly downstream of Raf, are frequently activated in carcinoid tumors (Tannapfel A, Vomschloss S, Karhoff D, Markwarth A, Hengge U R, Wittekind C, Arnold R, & Horsch D 2005 BRAF gene mutations are rare events in gastroenteropancreatic neuroendocrine tumors. Am. J. Clin. Pathol. 123 256-260; Karhoff D, SauerS, Schrader J, Arnold R, Fendrich V, Bartsch D K, & Horsch D 2007 Rap1/B-Raf signaling is activated in neuroendocrine tumors of the digestive tract and Raf kinase inhibition constitutes a putative therapeutic target. Neuroendocrinology 85 45-53; Perren A, Schmid S, Locher T, Saremaslani P, Bonvin C, Heitz P U, & Komminoth P 2004 BRAF and endocrine tumors: mutations are frequent in papillary thyroid carcinomas, rare in endocrine tumors of the gastrointestinal tract and not detected in other endocrine tumors. Endocr. Relat Cancer 11 855-860; Kunnimalaiyaan M & Chen H 2006 The Raf-1 pathway: a molecular target for treatment of select neuroendocrine tumors? Anticancer Drugs 17 139-142). The PI3K pathway is activated in carcinoid tumors from deletion of the tumor suppressor gene PTEN (phosphatase and tensin homologue). Loss of PTEN in neuroendocrine and carcinoid tumors, increases in frequency with the loss of differentiation in the tumor (Wang G G, Yao J C, Worah S, White J A, Luna R, Wu T T, Hamilton S R, & Rashid A 2005 Comparison of genetic alterations in neuroendocrine tumors: frequent loss of chromosome 18 in ileal carcinoid tumors. Mod. Pathol. 18 1079-1087), and loss of PTEN expression may represent an important step in the progression of neuroendocrine tumors (Wang L, Ignat A, & Axiotis C A 2002 Differential expression of the PTEN tumor suppressor protein in fetal and adult neuroendocrine tissues and tumors: progressive loss of PTEN expression in poorly differentiated neuroendocrine neoplasms. Appl. Immunohistochem. Mol. Morphol. 10 139-146).
Gastrointestinal and pulmonary carcinoid tumors are uncommon, but unfortunately are generally refractory to conventional cytotoxic chemotherapeutic and radiotherapeutic approaches. Many targeted therapeutic approach such as induction of Ras-mediated apoptosis by PKC delta inhibition, which selectively takes advantage of the very oncogenic mutations which contribute to the malignancy of the tumor, may have potential as a novel and selective therapeutic modality for these malignancies.